Qing Kai Ling Injection Rapid Determination Of Peptide Content And Impurities Of Protein, A Limited Check

Qingkailing injection is one of the modern Chinese medicine, which is developed by Beijing University of Chinese Medicine in basis of "Angongniuhuang", is a classic example of the secondary development of the famous prescriptions. The main component contains colalin, buffalo horn, mother-of-pearl, baicalin, gardenia, isatis root and honeysuckle, with the efficacy of clearing heat-toxin, inducing resuscitation, removing phlegm and dredging channels.It's mainly used for the treatment of fever coma, hemiplegia, acute and chronic hepatitis, epatitis b, upper respiratory tract infection, pneumonia and cerebrovascular disease clinically. It plays an irreplaceable important role in the treatment of urgent heavy fever.With its increasingly widespread application in clinical, the number of adverse reaction reported significantly increased, particularly the serious adverse reactions. It is facing unprecedented challenges. Qingkailing injection causes adverse reaction in the complex causes,one of the most important reasons is the protein and other impurities contained in the product or the oxidative degradation of active ingredients.This paper focused on the macromolecules of Qingkailing injection, then a rapid assaying method for the peptide active ingredient,a systemic separation method for the macromolecules and a limit test method for the protein impurity in the macromolecules was established to improve the quality standards.It will play an important role in improving the level of production quality control.Thesis research and the results are as follows:1, Bradford method combined with ultrafiltration centrifugation for determining protein and peptide content in traditional Chinese medicine injectionFristly, using centrifugal ultrafiltration to separate and concentrate protein and peptide with Qingkailing injection as the sample.Secondly, using coomassie blue staining to quantitatively determine the concentrated samples and then examine the estabilished methodologies.Experiment with different molecular weights selected according to different molecules interception by ultrafiltration centrifuge tube for molecular weight distribution of protein and peptide were studied together.Shenmai, Acanthopanax, Yin Zhi Huang, safflower, salvia and other fifteen traditional Chinese medicine injection were determined by this Limit test method, in orde to explore the area of this method in the applicability of traditional Chinese medicine injection.2, Exploratory study of the macromolecules'RP fingerprintIn the first chapter,we obtained Qingkailing injection of protein and peptide samples. Using Asahipak ODP 50 4E column to investigate the RP Fingerprints of samples on the phase column.3, Sephadex gel column used for establishing the macromolecules separation in Qingkailing injectionIn the second chapter, experiment results confirmed that the macromolecules samples preparated by ultrafiltration centrifugation remained in other small molecules. Use dextran gel filtration chromatography to remove Qingkailing injection of small molecules. Column, eluent, flow rate and the Sephadex model were respectively studied to establish the best separation conditions for the sample molecules.4, TSK gel column used in the limist test of protein impurities in Qingkailing injectionIn chapter three,the separation of macromolecules in Qingkailing injection was established. Use TSK gel column to analysis the samples prepared in chapter three. The linear relationship between molecular weight and retention time of five standard protein was studied,so as the linear relationship between peak area and content of standard protein. On this basis, a limited inspection standards was established for molecular weight and content of protein impurities in Qing Kai Ling Injection.The contents of the above experiment show that:(1) Bradford method combined with ultrafiltration centrifugation is practical in the test of the protein impurities in Chinese medicine injection, but there are a lot of restrictions in application, which need to expand their scope with further research.(2) the protein impurities limited screening method established by Sephadex chromatography with TSK gel chromatography limited olecular weight of impurities in protein content than 10KD shall not exceed lOppm. This method is applied in ten lots of Qingkailing injection, the results show that the method is stable, reproducible and free from the impact of changes in personnel and equipment, and limits are also very reasonable to be as a reference for Pharmacopoeia.